The ability of mammalian cell to sense the density of the cells around it plays an important role in cellular growth and differentiation. Without such an ability, a developing embryo would be unable to properly proportion its cells into different tissue types. Defects in cell density sensing may also be involved in cancer. Unfortunately, studying cell density sensing in mammalian cells is difficult due to their genetic intractability. However, the simple eukaryote Dictyostelium discoideum is an excellent genetic organism for the study of such a phenomenon. Dictyostelium cells monitor the density of starving cells by sensing the levels of CMF, a protein secreted by starving cells. When there is a sufficiently high density of starving cells, as indicated by a high extracellular concentration of CMF, the cells use relayed pulses of cAMP as a chemoattractant to allow them to aggregate and undergo development. cAMP binds to its receptor, cAR1, and activates a G protein signaling cascade culminating in the regulation of developmentally important genes. However, in the absence of CMF , while cAMP still binds to and activates its receptor, it does not activate downstream signaling components. CMF accomplishes the regulation by controlling the activity of the G protein associated with cAR1. This proposal describes experiments that will characterize the ability of CMF to regulate a G protein and delineate the proteins involved in this regulation. Specifically, the ability of CMF to affect the phosphorylation status of cAR1, the interaction cAR1 and its G protein, and the localization of known signaling proteins to the membrane will be examined. In addition, molecules that are involved in the regulation of the GTPase activity of the G protein will be identified.